Van Houten B, Gamper H, Hearst J E, Sancar A
Department of Biochemistry, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1988 Nov 15;263(32):16553-60.
Escherichia coli ABC excinuclease initiates the removal of dodecanucleotides from damaged DNA in an ATP-dependent reaction. Using a synthetic DNA fragment containing a psoralen adduct at a defined position we have investigated the interaction of the components of the enzyme with substrate by DNase I footprinting. We find that the UvrA subunit binds to DNA specifically in the absence of cofactors and that the binding affinity is stimulated about 4-fold by ATP and only marginally inhibited by ADP. The UvrA.DNA complexes formed in the absence of co-factors or in the presence of either ATP or ADP are remarkably similar. In contrast, adenosine 5'-O-(thiotriphosphate) increases nonspecific binding and completely abolishes the UvrA footprint. The UvrB subunit can associate with the UvrA subunit on DNA in the absence of ATP, but this ternary UvrA.UvrB.DNA complex is qualitatively different from that formed in the presence of ATP. The UvrC subunit elicits no additional change in the UvrA-UvrB footprint. Helicase II (UvrD protein) does not alter the UvrA-UvrB footprint but does appear to interact at the 5'-incision site of the postincision complex. DNA polymerase I fills in the excision gap in the presence or absence of helicase II and apparently releases the ABC excinuclease from the repaired DNA. Nearly 90% of the repair patches are 12 nucleotides long, and this length is not affected by helicase II. We see no evidence by DNase I footprinting for the formation of a multiprotein complex encompassing the UvrA, -B, -C, and -D proteins and DNA polymerase I.
大肠杆菌ABC核酸外切酶在ATP依赖反应中启动从受损DNA上去除十二核苷酸的过程。我们使用在特定位置含有补骨脂素加合物的合成DNA片段，通过DNase I足迹法研究了该酶各组分与底物的相互作用。我们发现，UvrA亚基在没有辅因子的情况下特异性结合DNA，ATP可使结合亲和力提高约4倍，而ADP仅轻微抑制结合。在没有辅因子或存在ATP或ADP的情况下形成的UvrA-DNA复合物非常相似。相比之下，腺苷5'-O-(硫代三磷酸)增加非特异性结合并完全消除UvrA足迹。在没有ATP的情况下，UvrB亚基可与DNA上的UvrA亚基结合，但这种三元UvrA-UvrB-DNA复合物与在ATP存在下形成的复合物在性质上不同。UvrC亚基不会使UvrA-UvrB足迹发生额外变化。解旋酶II（UvrD蛋白）不会改变UvrA-UvrB足迹，但似乎在切口后复合物的5'切口位点相互作用。DNA聚合酶I在有或没有解旋酶II的情况下填充切除缺口，并显然从修复的DNA上释放ABC核酸外切酶。近90%的修复片段长度为12个核苷酸，且这个长度不受解旋酶II影响。通过DNase I足迹法，我们没有发现形成包含UvrA、-B、-C、-D蛋白和DNA聚合酶I的多蛋白复合物的证据。
